Publication Date:
2015-11-14
Description:
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (〈1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Knight, Spencer C -- Xie, Liangqi -- Deng, Wulan -- Guglielmi, Benjamin -- Witkowsky, Lea B -- Bosanac, Lana -- Zhang, Elisa T -- El Beheiry, Mohamed -- Masson, Jean-Baptiste -- Dahan, Maxime -- Liu, Zhe -- Doudna, Jennifer A -- Tjian, Robert -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2015 Nov 13;350(6262):823-6. doi: 10.1126/science.aac6572.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Chemistry, University of California, Berkeley, CA, USA. ; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. ; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Transcriptional Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. ; Laboratoire Physico-Chimie Curie, Institut Curie, Centre National de la Recherche Scientifique UMR 168, Paris, France. ; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. ; Transcriptional Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Laboratoire Physico-Chimie Curie, Institut Curie, Centre National de la Recherche Scientifique UMR 168, Paris, France. ; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Transcriptional Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. liuz11@janelia.hhmi.org doudna@berkeley.edu jmlim@berkeley.edu. ; Department of Chemistry, University of California, Berkeley, CA, USA. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Innovative Genomics Initiative, University of California, Berkeley, CA, USA. liuz11@janelia.hhmi.org doudna@berkeley.edu jmlim@berkeley.edu. ; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Transcriptional Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. Li Ka Shing Biomedical and Health Sciences Center, University of California, Berkeley, CA, USA. liuz11@janelia.hhmi.org doudna@berkeley.edu jmlim@berkeley.edu.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26564855" target="_blank"〉PubMed〈/a〉
Keywords:
3T3 Cells
;
Animals
;
Bacterial Proteins/chemistry/*metabolism
;
*CRISPR-Cas Systems
;
Chromatin/chemistry/*metabolism/ultrastructure
;
Clustered Regularly Interspaced Short Palindromic Repeats
;
*DNA Cleavage
;
Endonucleases/chemistry/*metabolism
;
*Genetic Engineering
;
Genome
;
Mice
;
Single-Cell Analysis
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
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